Frances A. Edwards

Development of Rat CA1 Neurones in Acute Versus Organotypic Slices: Role of Experience in Synaptic Morphology and Activity

Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch‐clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations, at these corresponding stages. The most notable difference between organotypic and acute slices was a four‐ to five‐fold increase in the absolute frequency of glutamatergic (but not GABAergic) miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at a remarkably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

Synaptic P2X receptors.

Over the past two years, ATP has clearly been shown to act as a co-transmitter with GABA, glycine and probably glutamate in the central nervous system. Our understanding of the ATP-gated P2X receptors is progressing rapidly, and the pharmacology, stoichiometry and subunit combinations of heteropolymeric P2X channels has been substantially elucidated.

Effects of a naturally occurring neurosteroid on GABAA IPSCs during development in rat hippocampal or cerebellar slices.

1 The effects of the naturally occurring neurosteroid tetrahydrodeoxycorticosterone (THDOC) on GABAA receptor‐mediated miniature, spontaneous and evoked IPSCs was tested using patch‐clamp techniques in slices of hippocampus and cerebellum from rats at two developmental stages (≈10 and ≈20 days postnatal). The cells studied were hippocampal granule cells and cerebellar Purkinje and granule cells. 2 Most miniature GABAergic currents (mIPSCs) decayed with two exponentials and neurosteroids caused a ≈4‐fold increase in the decay time constant of the second exponential at the highest concentration used (2 μm). Similar effects were seen at high concentrations of THDOC (1‐2 μm) in all cell groups tested. No effects were seen on amplitude or rise time of mIPSCs. 3 The effects of THDOC (1 μm) were shown to be stereoselective and rapidly reversible, indicating that the neurosteroid binds to the GABAA receptor, rather than acting genomically. 4 At concentrations of THDOC likely to occur physiologically (50‐100 nm), the decay time of IPSCs was also enhanced (25‐50 %) in all cerebellar cell groups tested. In contrast, at 100 nm THDOC, seven of 11 hippocampal granule cells were sensitive from the 10 day group but the 20 day hippocampal granule cells showed no significant enhancement in the presence of these lower concentrations of THDOC. 5 The differences in sensitivity of hippocampal and cerebellar cells to THDOC are compared to data reported in the literature on regional development of expression of different receptor subunits in the brain and it is suggested that the progressive relative insensitivity of the 20 day hippocampal cells may depend on increasing expression of the δ subunit of the GABAA receptor and possibly an increase in the α4 subunit.

Properties of ATP receptor-mediated synaptic transmission in the rat medial habenula

The properties of central ATP-mediated synaptic currents were studied using whole-cell patch-clamp recording in rat medial habenula slices. Release was shown to be calcium dependent with a Hill coefficient of approximately 2. The voltage dependence of synaptic current amplitudes was approximately linear. Some reduction of the synaptic current amplitudes was observed at 10 mM extracellular calcium, suggesting calcium block/permeability of the channels. This was confirmed by observation of current-voltage reversal potentials in different calcium concentrations. We estimate that the channels underlying half the synapses showed a negligible calcium permeability. In the other four out of eight synapses the results suggest a very high calcium permeability with an estimated PCa/PCs of > 10. Thus, at -70 mV, in 1 mM calcium, more than 15% of the ATP-mediated synaptic current is estimated to be carried by calcium, but only at synapses with calcium-permeable channels. Net current through these synaptic channels is also controlled by the voltage dependence of synaptic current decay time constants (increasing e-fold for 158 mV depolarization) and by a strong dependence of transmitter release on the frequency of stimulation of the presynaptic neurone, with failure rates increasing 3-fold as stimulation rates were increased from 1 to 10 Hz.

ATP and glutamate are released from separate neurones in the rat medial habenula nucleus: frequency dependence and adenosine-mediated inhibition of release

1 ATP and glutamatergic synaptic currents were compared in slices of rat medial habenula nucleus using whole‐cell patch‐clamp techniques. 2 In most cells low voltage stimulation resulted in glutamatergic responses and not purinergic responses. In five cells where ATP currents could be stimulated with low voltages, wash out of glutamate antagonists did not reveal evoked glutamate currents. Spontaneous glutamate currents confirmed washout of antagonist. 3 Modulation of release probability of glutamate and ATP, assessed by changes in failure rate of synaptic currents, was compared under conditions of different stimulation frequencies and in the presence of adenosine agonists and antagonists. 4 ATP release, but not glutamate release, was shown to be modulated by increased stimulation frequency which resulted in inhibition of ATP release via A2‐like adenosine receptors. A1 receptors caused inhibition of both ATP and glutamate release. 5 Endogenous adenosine inhibited glutamate release via A1 receptors but only inhibited ATP release via A2‐like receptors. 6 Attempts to inhibit the degradation of ATP to adenosine did not alter the frequency dependence of the failure rate. 7 We conclude, from the direct demonstration and from the differences in pharmacology and frequency dependence of the modulation of release, that ATP and glutamate responses are due to release from separate neurones.

Corticosterone reduces dendritic complexity in developing hippocampal CA1 neurons

Although prolonged stress and corticosteroid exposure induce morphological changes in the hippocampal CA3 area, the adult CA1 area is quite resistant to such changes. Here we addressed the question whether elevated corticosteroid hormone levels change dendritic complexity in young, developing CA1 cells. In organotypic cultures (prepared from P5 rats) that were 14–21 days cultured in vitro, two doses of corticosterone (30 and 100 nM) were tested. Dendritic morphology of CA1 neurons was established by imaging neurons filled with the fluorescent dye Alexa. Application of 100 nM corticosterone for 20 minutes induced atrophy of the apical dendritic tree 1–4 hours later. Fractal analysis showed that total neuronal complexity was reduced twofold when compared with vehicle‐treated neurons. Exposing organotypic slices to 30 nM corticosterone reduced apical length in a more delayed manner: only neurons examined more than 2 hours after exposure to corticosterone showed atrophy of the apical dendritic tree. Neither dose of corticosterone affected the length of basal dendrites or spine density. Corticosterone was ineffective in changing morphology of the apical dendrites when tested in the presence of the glucocorticoid receptor antagonist RU38486. These results suggest that high physiological levels of corticosterone, via activation of the glucocorticoid receptor, can, during the course of only a few hours, reduce the dendritic complexity of CA1 pyramidal neurons in young, developing hippocampal tissue. These findings suggest that it is relevant to maintain plasma corticosterone levels low during hippocampal development.

The ducky2J Mutation in Cacna2d2 Results in Reduced Spontaneous Purkinje Cell Activity and Altered Gene Expression

The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a 2 bp deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C, du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

Long-term potentiation of glutamatergic synaptic transmission induced by activation of presynaptic P2Y receptors in the rat medial habenula nucleus.

A novel form of long‐term potentiation of glutamatergic synaptic transmission is described in the rat medial habenula nucleus. It occurs when uridine 5′‐triphosphate is bath applied at low micromolar concentrations and is prevented by Reactive Blue 2, suggesting that it is mediated by P2Y4 receptors. Uridine 5′‐diphosphate can also cause such a Reactive Blue 2‐sensitive potentiation, but at higher concentrations (200 µm), suggesting that this might also be an effect on the relatively uridine 5′‐diphosphate‐insensitive P2Y4 receptor. The potentiation is due to an increase in presynaptic release probability. It requires neither depolarization nor calcium influx postsynaptically and is thus probably non‐Hebbian. When potentiation due to low concentrations of uridine 5′‐triphosphate is inhibited in the presence of Reactive Blue 2, uridine 5′‐triphosphate causes instead a significant inhibition of glutamate release. We suggest that this inhibition may be mediated by a Reactive Blue 2‐insensitive P2Y2‐like receptor. At higher concentrations of uridine 5′‐triphosphate (200 µm), the inhibitory effect dominates such that even in the absence of Reactive Blue 2 no potentiation is seen.

GABA release by basket cells onto Purkinje cells, in rat cerebellar slices, is directly controlled by presynaptic purinergic receptors, modulating Ca2+ influx.

In many brain regions, Ca(2+) influx through presynaptic P2X receptors influences GABA release from interneurones. In patch-clamp recordings of Purkinje cells (PCs) in rat cerebellar slices, broad spectrum P2 receptor antagonists, PPADS (30microM) or suramin (12microM), result in a decreased amplitude and increased failure rate of minimal evoked GABAergic synaptic currents from basket cells. The effect is mimicked by desensitizing P2X1/3-containing receptors with alpha,beta-methylene ATP. This suggests presynaptic facilitation of GABA release via P2XR-mediated Ca(2+) influx activated by endogenously released ATP. In contrast, activation of P2Y4 receptors (using UTP, 30microM, but not P2Y1 or P2Y6 receptor ligands) results in inhibition of GABA release. Immunological studies reveal the presence of most known P2Rs in >or=20% of GABAergic terminals in the cerebellum. P2X3 receptors and P2Y4 receptors occur in approximately 60% and 50% of GABAergic synaptosomes respectively and are localized presynaptically. Previous studies report that PC output is also influenced by postsynaptic purinergic receptors located on both PCs and interneurones. The high Ca(2+) permeability of the P2X receptor and the ability of ATP to influence intracellular Ca(2+) levels via P2Y receptor-mediated intracellular pathways make ATP the ideal transmitter for the multisite bidirectional modulation of the cerebellar cortical neuronal network.

The function of A2 adenosine receptors in the mammalian brain: evidence for inhibition vs. enhancement of voltage gated calcium channels and neurotransmitter release.

Publisher Summary This chapter discusses the results in the context of previous reports on the role of A 2 receptors in synaptic transmission in the mammalian brain, and concludes that there are few cases in which A 2 receptors have been shown to be excitatory in directly increasing the release of a specific central nervous system (CNS) neurotransmitter. Although in several studies the overall effect of the activation of A 2 receptors has been to increase network excitability, most of these results would be compatible with an indirect effect through inhibition of γ-aminobutyric acid (GABA) release. GABA release tends to be inhibited by activation of A 2 receptors that would be consistent with the inhibition of N- and L-type calcium channels and also with the frequent observation of increased excitability of neuronal networks. Activation of A 2 receptors has been shown to result in inhibition of both calcium channels and GABAergic transmission. Presumably, the localization of A 2 receptors on GABAergic terminals results in inhibition of calcium channels and inhibition of GABA release.